Cholera is an ancient disease that results in significant morbidity and mortality. The broad aim and focus of this project is to understand the pathogenesis of Vibrio cholera pathogenicity island phage (VPIphage) and its genes in the emergence, virulence, and spread of epidemic cholera. Cholera is traditionally caused by V. cholerae serogroup 01 strains which are divided into two biotypes, classical and El Tor. Although recent emergence of V. cholerae 0139 Bengal has demonstrated that virulent non-01 strains can occur, this strain appears to have evolved from an existing toxigenic 01 strain by substitution of 01 antigen genes to 0139. Thus, the factors involved in the emergence of virulent V. cholerae strains and the epidemic potential of non-01 serogroups are not well understood. Toxigenic V. cholerae appears to emerge via a multi-step process following the sequential infection of two bacteriophages, initially by VPIphage (whose genome represents a large pathogenicity island) then CTXphage (encoding cholera toxin) which uses VPIphage as its receptor. The VPIphage encodes genes which function as virulence factors, genes that regulate virulence factor expression, and a number of open reading frames (orfs) of unknown function. The VPIphage-encoded tcpA gene was thought to encode the major subunit of the toxin co-regulated pilus (TCP) "type IV" pilus, but it now appears that the VPIphage itself is the major V. cholerae colonization factor and that TcpA is the major structural coat protein of VPIphage. The central hypothesis of this proposal is that the VPIphage is critical for the emergence, pathogenesis, and spread of epidemic cholera as 1) the VPIphage is essential for colonization; 2) VPIphage encodes proteins which act as the CTXphage receptor; 3) VPIphage encodes genes which regulate virulence; d) VPIphage encodes genes for transfer of the VPIphage between strains. The specific aims of this proposal are to 1) test the virulence of the VPI in a non-pathogenic host background; 2) analyze the function of cryptic genes on the VPI; 3) to characterize the structure and regulation of a potential VPI-encoded regulon. It is hoped that the proposed experiments will increase the knowledge of the mechanisms leading to the emergence of epidemic strains of cholera and or the emergence of other pathogens that have pathogenicity islands and type IV pili.